Hendra virus and the closely related Nipah virus are zoonotic paramyxoviruses with high mortality rates in humans, and both have been placed on the NIAID priority pathogens list. The fusion (F) protein of Hendra virus promotes both virus-cell and cell-cell membrane fusion, thereby mediating a critical first step in viral infection. This important viral protein undergoes proteolytic cleavage of the precursor form, F0, to the disulfide-linked form, F1 and F2, a process that has been shown to be required for fusogenic activity of all paramyxovirus F proteins characterized to date. It is clear that the cellular secretory protease furin, known to cleave other paramxyovirus F proteins, is not involved in proteolytic activation of the Hendra F protein. At present, neither the cellular localization of this crucial cleavage event or the identity of the protease involved is known. The long-term objective of this proposed research is to understand the processing of the Hendra F protein, and to identify host cell factors involved in these events. The specific hypothesis to be tested is that the protease involved in cleavage of the Hendra F protein represents a novel class of cellular proteases that could serve as a target for anti-viral therapies. In our preliminary results, we demonstrate that proteolytic activation occurs in either secretory vesicles budding from the trans-Golgi network or at the cell surface. In addition, we find that proteolytic processing is not affected by Ca2+ levels, but is extremely sensitive to changes in intracellular pH. The experiments in Aim 1 will identify the cellular location of proteolytic processing of the Hendra F protein and the amino acid sequence requirements for cleavage. The research proposed in Aim 2 is directed towards purification of the protease responsible for proteolytic processing of the Hendra F protein, with subsequent identification using N-terminal sequencing or proteomic techniques. This research will provide important information about basic processes within the secretory pathway. In addition, identification of the enzyme(s) involved in the proteolytic cleavage of the Hendra and Nipah F proteins will set the stage for development of new antiviral therapies for these recently emerged paramyxoviruses.